JP7054922B2 - Solid phase peptide synthesis methods and related systems - Google Patents

Description

Related Applications This application claims priority to US Provisional Application No. 62 / 220,232 filed on September 17, 2015 under Section 119 (e) of the US Patent Act. , The content of this provisional application is incorporated herein by reference in its entirety for all purposes.

Technical Fields The methods and systems for performing solid phase peptide synthesis are outlined.

Background Solid phase peptide synthesis is a process used to chemically synthesize peptides on solid supports. In solid phase peptide synthesis, the amino acid or peptide is usually attached to the solid support via the C-terminus. New amino acids are added to the bound amino acids or peptides via a coupling reaction. Protecting groups are typically used because of the potential for unintended reactions. Solid-phase peptide synthesis has become a standard practice for chemical peptide synthesis. The widespread usefulness of solid phase peptide synthesis has been demonstrated by the commercial success of automated solid phase peptide synthesis. Although solid phase peptide synthesis has been used for over 30 years, no automated solid phase peptide synthesizer has yet been developed that provides a high degree of control over individual coupling reactions and / or minimizes side reactions. Therefore, improved processes and systems are needed.

Abstract A solid phase peptide synthesis method and related systems are outlined. Certain embodiments relate to systems and methods for activating amino acids. In some embodiments, the activation reagents can be combined to reduce the amount of side reactions and increase the yield. The subject matter of the present invention, in some cases, includes interrelated products, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

In one set of embodiments, methods are provided. In one embodiment, the method of manipulating the peptide synthesis system is to flow a first fluid stream containing amino acids, a second fluid stream containing a base, and a first and second fluid stream in a mixed region. It involves merging to form a mixed fluid flow, as well as flowing the mixed fluid flow through the reactor. In such an embodiment, the molar ratio of amino acids to bases in the mixed fluid stream in the mixed region is within 10% of the molar ratio of amino acids to bases in the reactor.

In another embodiment, the method of manipulating the peptide synthesis system is to flow a first fluid stream containing an activator, a second fluid stream containing a base, a first and a second fluid stream. It involves merging in a mixing region to form a mixed fluid flow, as well as flowing the mixed fluid flow into a reactor. In such an embodiment, the molar ratio of base to activator in the mixed fluid stream in the mixed region is within 10% of the molar ratio of base to activator in the reactor.

In one embodiment, the method of manipulating the peptide synthesis system is to flow a first fluid stream containing amino acids, a second fluid stream containing a base, and a first and second fluid stream in a mixed region. It involves merging to form a mixed fluid flow, as well as flowing the mixed fluid flow into a reactor. In such embodiments, the molar ratio of amino acids to bases in the mixed fluid stream in the mixed region is the reaction over a period of time starting from the time when at least one of the amino acids and bases first reaches the reactor. It is within 10% of the molar ratio of amino acids to bases in the vessel.

In another embodiment, the method of manipulating the peptide synthesis system is to flow a first fluid stream containing an activator, a second fluid stream containing a base, a first and a second fluid stream. It involves merging in a mixing region to form a mixed fluid flow, as well as flowing the mixed fluid flow into a reactor. In such an embodiment, the mole of the base and the activator in the mixed fluid stream in the mixed region over a period of time starting from the time when at least one of the base and the activator first reaches the reactor. The ratio is within 10% of the molar ratio of base to activator in the reactor.

In one embodiment, the method of initiating the operation of the peptide synthesis system is to flow a first fluid stream containing amino acids into the mixed region, a second fluid flow containing bases into the mixed region, first and first. Combining the fluid streams of 2 in the mixing region, including forming a mixed fluid stream with a leading edge, as well as flowing the mixed fluid stream into the reactor. In such an embodiment, the amino acid to base molar ratio measured at the leading edge as it enters the reactor is 10 of the amino acid to base molar ratio in the mixed fluid stream in the mixed region. Within%.

In another embodiment, the method of initiating the operation of the peptide synthesis system is to flow a first fluid flow mixing region containing an activator, a second fluid flow containing a base to the mixing region, first. And the confluence of the second fluid flow in the mixing region, which comprises forming a mixed fluid flow with a leading edge and flowing the mixed fluid flow into the reactor. In such an embodiment, the molar ratio of base to activator measured at the leading edge as it enters the reactor is the base and activator in the mixed fluid flow in the mixed region. It is within 10% of the molar ratio.

In one embodiment, the method of initiating the operation of the peptide synthesis system is to initiate a first fluid flow containing amino acids from the first reagent reservoir into the mixing region, a second fluid flow containing bases. To start flowing from the reagent reservoir to the mixing region so that the first fluid flow and the second fluid flow reach the mixing region within about 10 ms of each other, the first and second fluids. It involves merging the streams in a mixing region to form a mixed fluid stream, as well as flowing the mixed fluid stream into a reactor.

In another embodiment, the method of initiating the operation of the peptide synthesis system is to initiate a first fluid flow containing an activator from the first reagent reservoir into the mixing region, a second fluid flow containing a base. Is to start flowing from the second reagent reservoir to the mixing region so that the first fluid flow and the second fluid flow reach the mixing region within about 10 ms of each other. It involves merging the fluid streams of 2 in the mixing region to form a mixed fluid stream, as well as flowing the mixed fluid stream into a reactor.

In one embodiment, the method of manipulating the peptide synthesis system is to combine a first fluid flow containing amino acids and a second fluid flow containing bases at the junction to form a mixed fluid flow, as well. Flowing the mixed fluid flow from the junction to the reactor, including introducing the mixed fluid flow into the reactor, with a residence time of the mixed fluid flow from the junction to the reactor of at least about 0.1 seconds. It takes less than about 30 seconds and the molar ratio of amino acids to bases in the mixed fluid flow varies by less than 10% from the formation of the mixed fluid flow to the introduction of the mixed fluid flow into the reactor.

In another embodiment, the method of initiating the operation of the peptide synthesis system is to flow a first fluid stream containing amino acids into the mixed region, a second fluid flow containing bases into the mixed region, first and. Combining a second fluid flow in the mixing region, including forming a mixed fluid flow with a leading edge, as well as flowing the mixed fluid flow into the reactor, when the leading edge enters the reactor. The molar ratio of amino acids to bases measured at the leading edge is at least about 10 ms after the leading edge enters the reactor with the amino acids in the mixed fluid flow at the entrance to the reactor. It is within 10% of the molar ratio with the base.

In one embodiment, the method of initiating the operation of the peptide synthesis system is to flow a first fluid stream containing an activator into the mixed region, a second fluid flow containing a base into the mixed region, first. And the confluence of the second fluid flow in the mixing region, including forming a mixed fluid flow with a leading edge, and allowing the mixed fluid flow to flow into the reactor, with the leading edge entering the reactor. The molar ratio of activator to base, sometimes measured at the leading edge, is in the mixed fluid flow at the entrance to the reactor at least about 10 ms after the leading edge enters the reactor. It is within 10% of the molar ratio of the activator and the base.

Other advantages and novel features of the invention will be apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. If the specification and the documents incorporated by reference contain conflicting and / or contradictory disclosures, the specification shall prevail.

Non-limiting embodiments of the present invention are described as examples with reference to the accompanying drawings, which are schematic and not drawn to scale. In the drawings, each of the same or nearly identical components illustrated is typically represented by a single reference numeral. For clarity purposes, not all components are labeled with all components and the invention is not required to be illustrated to allow one of ordinary skill in the art to understand the invention. Not all components of each embodiment of the above are shown.
In the embodiment of the present invention, for example, the following items are provided.
(Item 1)
A way to initiate the operation of a peptide synthesis system,
Flowing a first fluid stream containing an activator into the mixing region,
Flowing a second fluid stream containing a base into the mixed region,
By merging the first and second fluid flows in a mixed region, forming a mixed fluid flow with a leading edge, and
The molar ratio of the activator to the base measured at the leading edge when the leading edge enters the reactor, including flowing the mixed fluid stream through the reactor, is such that the leading edge is said. A method within 10% of the molar ratio of the activator to the base in the mixed fluid flow at the entrance to the reactor at least about 10 ms after entry into the reactor. ..
(Item 2)
A way to initiate the operation of a peptide synthesis system,
Flowing a first fluid stream containing amino acids into the mixed region,
Flowing a second fluid stream containing a base into the mixed region,
By merging the first and second fluid flows in a mixed region, forming a mixed fluid flow with a leading edge, and
The molar ratio of the amino acid to the base measured at the leading edge when the leading edge enters the reactor comprises flowing the mixed fluid stream through the reactor, with the leading edge being the reactor. The method, which is within 10% of the molar ratio of the amino acid to the base in the mixed fluid flow at the entrance to the reactor at least about 10 ms after entry into the reactor.
(Item 3)
The molar ratio of the amino acid to the base measured at the leading edge when the leading edge enters the reactor is at least about 50 ms after the leading edge enters the reactor, at least about 100 ms. After, or at least about 1 second later, to any of the above items, which is within 10% of the molar ratio of the amino acid to the base in the mixed fluid flow at the entrance to the reactor. The method described.
(Item 4)
When the molar ratio of the activator to the base measured at the leading edge as it exits the mixed region is at least about 10 ms after the leading edge exits the mixed region. The method according to any one of the above items, wherein the mixed fluid flow is within 10% of the molar ratio of the activator to the base in the mixed fluid flow as it exits the mixed region. ..
(Item 5)
A way to initiate the operation of a peptide synthesis system,
Initiating a first fluid stream containing amino acids from the first reagent reservoir into the mixing region,
A second fluid flow containing a base is started to flow from the second reagent reservoir into the mixed region, and the first fluid flow and the second fluid flow are in the mixed region within about 10 ms of each other. To reach,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
Flowing the mixed fluid flow through the reactor
How to include.
(Item 6)
A way to initiate the operation of a peptide synthesis system,
Initiating a first fluid stream containing an activator from the first reagent reservoir into the mixing region,
A second fluid flow containing a base is started to flow from the second reagent reservoir into the mixed region, and the first fluid flow and the second fluid flow are in the mixed region within about 10 ms of each other. To reach,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
Flowing the mixed fluid flow through the reactor
How to include.
(Item 7)
The method according to any of the above items, comprising flowing a third fluid stream containing an activator.
(Item 8)
The method according to any of the above items, comprising flowing a third fluid stream containing an amino acid.
(Item 9)
The method according to any of the above items, further comprising merging the first, second, and third fluid streams in the mixing region.
(Item 10)
The method according to any of the above items, comprising flowing a fourth fluid stream, comprising an additive selected from the group consisting of chaotropic salts, co-solvents, and surfactants.
(Item 11)
The method according to any of the above items, further comprising merging the first, second, third, and fourth fluid streams in the mixing region.
(Item 12)
The molar ratio of the amino acid to the base, measured at the leading edge as it enters the reactor, is the base and activator in the mixed fluid flow in the mixed region. The method according to any one of the above items, which is within 10% of the molar ratio of.
(Item 13)
The molar ratio of the base to the activator, measured at the leading edge as it enters the reactor, is the activation of the base in the mixed fluid stream in the mixed region. The method according to any of the above items, which is within 10% of the molar ratio with the agent.
(Item 14)
The molar ratio of the amino acid to the base, measured at the leading edge when the leading edge enters the reactor, is at least about 10 ms after the leading edge enters the reactor. The method according to any one of the above items, which is within 10% of the molar ratio of the activator to the base in the mixed fluid flow at the entrance to the reactor.
(Item 15)
The molar ratio of the base to the activator, measured at the leading edge when the leading edge enters the reactor, is at least about 10 ms after the leading edge enters the reactor. The method according to any of the above items, which is within 10% of the molar ratio of the activator to the base in the mixed fluid flow at the entrance to the reactor at a time point.
(Item 16)
The method according to any one of the above items, wherein the molar ratio of amino acids to bases in the mixed fluid flow is higher than about 1: 1.
(Item 17)
The method according to any one of the above items, wherein the activator is selected from the group consisting of carbodiimide, guanidinium salt, phosphonium salt, and uronium salt.
(Item 18)
The method according to any of the above items, wherein the leading edge of the first fluid flow reaches the mixed region within 10 ms of the leading edge of the second fluid flow.
(Item 19)
A method of manipulating a peptide synthesis system,
Combining a first fluid flow containing amino acids and a second fluid flow containing bases at a junction to form a mixed fluid flow, and
Including flowing the mixed fluid flow from the junction to the reactor and introducing the mixed fluid flow into the reactor, the residence time of the mixed fluid flow from the junction to the reactor is at least about about. 0.1 seconds and less than about 30 seconds
A method in which the molar ratio of the amino acid to the base in the mixed fluid stream varies by 10% or less from the formation of the mixed fluid stream to the introduction of the mixed fluid stream into the reactor.
(Item 20)
A method of manipulating a peptide synthesis system,
Flowing a first fluid stream containing amino acids,
Flowing a second fluid stream containing bases,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
Including flowing the mixed fluid flow through the reactor, the molar ratio of the amino acid to the base in the mixed fluid flow in the mixed region is the molar ratio of the amino acid to the base in the reactor. A method that is within 10%.
(Item 21)
A method of manipulating a peptide synthesis system,
Flowing a first fluid stream containing an activator,
Flowing a second fluid stream containing bases,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
The molar ratio of the base to the activator in the mixed fluid flow in the mixed region comprises flowing the mixed fluid stream through the reactor, with the base and the activator in the reactor. The method, which is within 10% of the molar ratio of.
(Item 22)
A method of manipulating a peptide synthesis system,
Flowing a first fluid stream containing amino acids,
Flowing a second fluid stream containing bases,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
The said in the mixed fluid stream in the mixed region over a period of time starting from the time when at least one of the amino acids and bases first reaches the reactor, comprising flowing the mixed fluid stream through the reactor. A method in which the molar ratio of an amino acid to the base is within 10% of the molar ratio of the amino acid to the base in the reactor.
(Item 23)
A method of manipulating a peptide synthesis system,
Flowing a first fluid stream containing an activator,
Flowing a second fluid stream containing bases,
By merging the first and second fluid flows in the mixing region, forming a mixed fluid flow, and
In the mixed fluid flow in the mixed region over a period of time starting from the time when at least one of the base and the activator first reaches the reactor, comprising flowing the mixed fluid flow through the reactor. The method in which the molar ratio of the base to the activator is within 10% of the molar ratio of the base to the activator in the reactor.
(Item 24)
A way to initiate the operation of a peptide synthesis system,
Flowing a first fluid stream containing amino acids into the mixed region,
Flowing a second fluid stream containing a base into the mixed region,
By merging the first and second fluid flows in a mixed region, forming a mixed fluid flow with a leading edge, and
The molar ratio of the amino acid to the base measured when the leading edge enters the reactor comprises flowing the mixed fluid flow through the reactor, and the molar ratio of the amino acid to the base in the mixed fluid flow in the mixed region. The method, which is within 10% of the molar ratio of the amino acid to the base.
(Item 25)
A way to initiate the operation of a peptide synthesis system,
Flowing a first fluid stream containing an activator into the mixing region,
Flowing a second fluid stream containing a base into the mixed region,
By merging the first and second fluid flows in a mixed region, forming a mixed fluid flow with a leading edge, and
The molar ratio of the base to the activator measured when the leading edge enters the reactor comprises flowing the mixed fluid flow through the reactor, and the mixed fluid flow in the mixed region. A method in which the molar ratio of the base to the activator is within 10%.
(Item 26)
The method according to any of the above items, wherein the base is a Lewis base.
(Item 27)
The method according to any of the above items, wherein the base is a non-nucleophilic base.
(Item 28)
The method according to any of the above items, wherein the mixed fluid stream is not exposed to heat from a heat source before reaching the reactor.
(Item 29)
The method according to any of the above items, wherein the mixed fluid flow is not exposed to heat from a heat source between the mixing region and the entrance to the reactor.
(Item 30)
The method according to any of the above items, wherein the temperature of the mixed fluid flow is within 10 ° C. of the temperature of the leading edge at the inlet of the reactor.
(Item 31)
The method according to any of the above items, wherein the temperature of the leading edge changes by less than 10 ° C. from the mixing region to the inlet of the reactor.
(Item 32)
The method according to any of the above items, wherein the reactor does not contain a plurality of amino acids immobilized on a solid support.
(Item 33)
The method according to any of the above items, wherein the reactor does not contain a plurality of peptides immobilized on a solid support.
(Item 34)
The method of any of the above items, wherein the reactor comprises a plurality of peptides immobilized on a solid support.
(Item 35)
The method according to any of the above items, comprising exposing the amino acid to the immobilized peptide, wherein at least a portion of the amino acid is bound to the immobilized peptide to form a long peptide. ..
(Item 36)
The method according to any of the above items, wherein more than about 99% of each of the immobilized peptides will be bound to a single amino acid molecule during the exposure step, respectively.
(Item 37)
The method of any of the above items, wherein the solid support is housed in a packed column and / or a fluidized bed.
(Item 38)
The method according to any of the above items, wherein the solid support contains a resin.
(Item 39)
The method according to any one of the above items, wherein the solid support comprises a microporous polystyrene resin, a microporous polyethylene glycol resin, and / or a microporous copolymer resin.
(Item 40)
The method according to any of the above items, further comprising providing a plurality of peptides comprising a protecting group, each peptide immobilized on a solid support.
(Item 41)
The exposure of the deprotecting reagent to the immobilized peptide further comprises removing the protecting group from at least a portion of the immobilized peptide and removing at least a portion of the deprotecting reagent. The method described in any of the items.
(Item 42)
Exposing the activated amino acid to the immobilized peptide further comprises forming a newly bound amino acid residue by binding at least a portion of the activated amino acid to the immobilized peptide. The method according to any of the above items.
(Item 43)
The method according to any of the above items, further comprising removing at least a portion of the activated amino acid that does not bind to the immobilized peptide.
(Item 44)
The method according to any of the above items, wherein the protecting group comprises a fluorenylmethyloxycarbonyl protecting group.
(Item 45)
The method according to any of the above items, wherein the protecting group comprises a tert-butyloxycarbonyl protecting group.

FIG. 1 is a schematic diagram of solid phase peptide synthesis according to a set of embodiments. FIG. 1 is a schematic diagram of solid phase peptide synthesis according to a set of embodiments. FIG. 2A is a schematic diagram of a system for performing peptide synthesis according to a set of embodiments. FIG. 2B is a schematic diagram of a system for performing peptide synthesis according to a set of embodiments. FIG. 3A is an exemplary schematic of a peptide synthesis system according to a particular embodiment. FIG. 3B is a chromatogram of the synthesized peptide according to a particular embodiment. FIG. 3C is a concentration profile for two activation reagents according to a particular embodiment. FIG. 4A is a chromatogram of the synthesized peptide according to a set of embodiments. FIG. 4B is a graph of the percentage of peptides synthesized at various flow rates to the product of the identified product, according to a set of embodiments. FIG. 5A is a photograph of an automated fluid solid phase synthesizer according to a set of embodiments. FIG. 5B is a cycle diagram of peptide synthesis according to a particular embodiment. FIG. 5C is an LC-MS chromatogram for a crude product of acyl carrier protein (65-74) synthesis according to a set of embodiments. FIG. 5D is a UV absorbance spectrum for one coupling and deprotection cycle according to a set of embodiments. FIG. 6A is an LC-MS chromatograph for Growth Hormone Releasing Hormone (GHRH) synthesized via different methods according to a set of embodiments. FIG. 6B is an LC-MS chromatograph for insulin B chains synthesized using different methods according to a set of embodiments. FIG. 6C is a plot of Fmoc deprotected UV data for each cycle of synthesis for GHRH and insulin B chains according to a set of embodiments. FIG. 7A is a diagram of a heated portion of an automated fluid peptide synthesizer according to a set of embodiments. FIG. 7B shows a representative sample from fluid synthesis using Method B according to a set of embodiments (top) and a 50/50 mixture of authentic Cys diastereomers (bottom) of the model peptide GCF. Diastereomer analysis. FIG. 7C is a graph of the percentage of Cys diastereomer formation as a function of flow rate (ml / min) using method B according to a set of embodiments. FIG. 7D shows a representative sample from fluid synthesis using Method B according to a set of embodiments (top) and a 50/50 mixture of authentic Cys diastereomers (bottom) of the model peptide FHL. Diastereomer analysis. FIG. 7E is a graph of the percentage of histidine diastereomeric formation as a function of flow rate (ml / min).

Detailed Description Methods and systems for solid phase peptide synthesis are provided. Solid phase peptide synthesis is a known process in which amino acid residues are added to a peptide immobilized on a solid support. New amino acid residues are added via a coupling reaction between the activated amino acid and the amino acid residue of the immobilized peptide. Amino acids may be activated using, for example, bases and activators. Certain concepts of the invention described herein relate to methods and systems for activating amino acids. These systems and methods can allow fewer side reactions and higher yields compared to traditional activation techniques, and are costly and / or residue-by-residue cups without the need for complex processes. It may allow customization of the ring reaction.

A non-limiting outline of the solid phase peptide synthesis method is shown in FIG. In some embodiments, the solid phase synthesis method may utilize the solid support 10. Peptide 15 may be bound such that each peptide is immobilized on a solid support. For example, the peptide may be attached to a solid support via its C-terminus 20, thereby immobilizing the peptide. In certain embodiments, peptide 15 may contain, for example, a protecting group 25 on the N-terminus 30 of the peptide. In some embodiments, the side chain 35 of the amino acid residue in the peptide may contain a protecting group as shown in FIG. In some embodiments, the process of adding an amino acid residue to the immobilized peptide deprotects at least a portion of the N-terminal protecting group as shown by arrow 40 and free N as shown in FIG. Includes forming the terminal 45.

In some embodiments, the free N-terminus may be exposed to an amino acid, allowing at least a portion of the free N-terminus to be coupled to the C-terminus of the amino acid, as a result of which is indicated by the arrow 50. As such, an amide bond is formed and a newly bound amino acid residue is added to the immobilized peptide. In certain embodiments, amino acid 55 may be activated prior to exposure to the free N-terminus 45, as indicated by arrow 70. Amino acid activation may accelerate the coupling reaction such that the yield of the coupling reaction is relatively high (eg, greater than or equal to about 98%).

In general, activated amino acids may be formed using any suitable reagent. In certain embodiments, the activating amino acid may be formed using activator 60 and base 65, as shown in FIG. 1 and as indicated by arrow 70. In some such embodiments, the activating amino acid does not have to be purified prior to exposure to the immobilized peptide. In such cases, the immobilized peptide may be exposed to at least a portion of unreacted activators, bases, and / or amino acids, some of which may adversely affect peptide synthesis. For example, an unreacted uranium or guanidinium activator may undergo a coupling reaction with at least a portion of the free N-terminus to prevent further addition of amino acid residues. Thus, in some embodiments, precise control of stoichiometric ratios of activators (eg, activators, bases, and / or amino acids) prevents side reactions and thereby increases overall yield. Is needed.

In some conventional systems that control stoichiometric ratios, the activation reagent is mixed for an extended period of time and then exposed to the immobilized peptide and the mixture is stored in the system. In certain embodiments, storing certain activation reagents together can result in undesired side reactions prior to exposure to the immobilized peptide. For example, storage of amino acids with bases can result in degradation, polymerization, removal of protecting groups, and / or epimerization of amino acids. In some cases, the yield and kinetics of the coupling reaction will be adversely affected. Certain conventional systems have attempted to address this challenge by mixing activation reagents in the presence of immobilized peptides. However, this method can result in slower reaction rates, cleavage of peptidyl chains, and ultimately lower yields.

Certain methods and systems of the invention described herein have little or no undesired effect on adverse side reactions and / or yields (eg, of the coupling reaction as a whole). Allows stoichiometric control of activation reagents. In some embodiments, the one or more activation reagents may be stored separately from another activation reagent. For example, amino acids and / or activators may be stored separately from the base (eg, in reagent reservoirs). Two or more activation reagents may be mixed before reaching the reactor but within a short time to reach the reactor, the reactor being desired for activation. As a mixture with a ratio, it will first be exposed to two or more activators. In some such embodiments, a first fluid stream comprising a first activating reagent (eg, an amino acid, an activator) and a second containing a second activating reagent (eg, a base). The fluid flow may be merged at the mixing region (eg, the junction) to form a mixed fluid with a leading edge, which flows into the reactor. In some such cases, the molar ratio of the two or more activating reagents in the mixed region (eg, bases and amino acids, bases and activators) is two or more in the reactor. Is also within 10% (eg, 5%) of the molar ratio of many activators. In certain embodiments, the flow of the first and second fluid flows is such that the leading edge of the first fluid flow and the leading edge of the second fluid flow are within about 10 ms of each other (eg, substantially simultaneously). It may be controlled at the start of the fluid flow to arrive. In some embodiments, the first and second fluid streams are two or more activation reagents (eg, bases) in the mixed region as the leading edge of the mixed fluid enters the reactor. And amino acids, bases and activators) can be merged so that the molar ratio of the two or more activators in the reactor is within 10% (eg, 5%) of the molar ratio of the activator. good. In some embodiments, the individual activation reagents do not have to be introduced into the reactor due to merging in the mixed region.

Schematic representations of exemplary systems 80 and 200 that can be used to perform certain activation methods described herein are shown in FIGS. 2A and 2B. The systems and methods described herein (eg, system 80 in FIG. 2A, system 200 in FIG. 2B) are capable of performing flow-based synthesis (in many traditional solid phase peptide synthesis systems). In contrast to the batch-based synthesis used). In some such embodiments, continuous peptide synthesis is possible in which the fluid (in any form) is transported substantially continuously to the immobilized peptide within the reactor. For example, reagents and rinsing fluids may, in certain embodiments, be transported alternately and continuously onto the immobilized peptide.

For example, in some embodiments, the system may include a tank such as a reactor containing peptides and / amino acids immobilized on a solid support. For example, as shown in FIG. 2A, peptide 85 may be immobilized on a solid support 90. The solid support 90 may be placed in a tank such as a reactor 95. In some embodiments, as shown in FIG. 2A, a plurality of reagent reservoirs may be located upstream of the reactor 95 and fluidly connected. In some embodiments, the reagent reservoir 100 contains amino acids. In some cases, the reagent reservoir 105 contains a base (eg, diisopropylethylamine). In certain embodiments, the reagent reservoir 110 contains an activator such as a carbodiimide, guanidinium salt, phosphonium salt, or uronium salt. In some embodiments, the system 80 may include an optional reagent reservoir 120. In some cases, the reagent reservoir 120 may contain one or more additives such as chaotropic salts, co-solvents, and / or surfactants. In certain embodiments, the system 80 may include a second optional reagent reservoir 125. In some cases, the reagent reservoir 125 may contain a deprotecting reagent such as piperidine or trifluoroacetic acid, or may contain a solvent such as dimethylformamide (DMF) which may be used in the washing step, for example. ..

In some embodiments, the system comprises a tank, such as a reactor, configured to facilitate and / or facilitate one or more chemical reactions between molecules. For example, as shown in FIG. 2B, the system 200 has one or more chemical reactions between certain reagents and / or their reaction products, for example by modulating the reaction rate and / or reaction time. May include a reactor 205 configured to facilitate and / or facilitate. For example, the reactor 205 controls the temperature profile of the fluid flow in the reactor so that one or more temperature-dependent reaction rates are modulated (eg, increased, maintained, and / or decreased). ) To be able to achieve the desired reaction rate (s), reaction product (s), amount of reaction product (s), and / or reaction yield (s). It may be configured. In some embodiments, multiple reagent reservoirs (eg, 210, 215, 220) may be located upstream of the reactor 205 and fluidly connected, as shown in FIG. 2B. For example, a reagent reservoir 210 (eg, containing amino acids), a reagent reservoir 215 (eg, containing a base), and / or a reagent reservoir 220 (eg, containing an activator) can be used in the reactor 205. It may be located upstream of and fluidly connected. In some embodiments, the system 200 is located upstream of the reactor 205 and is fluid-connected to the reagent reservoir 225 (eg, containing additives) and / or the reagent reservoir 230 (eg, eg). It may include one or more optional reagent reservoirs, such as (containing deprotecting reagents).

In some embodiments, the reactor 205 is located between reagents from one or more reservoirs located upstream of the reactor 205, between reagent reaction products and reagents, and / or reagent reaction generation. It may be configured to facilitate and / or facilitate chemical reactions between objects. In certain embodiments, the reactor 205 may facilitate and / or facilitate a chemical reaction between two or more reagents from a reservoir located upstream of the reactor 205. .. For example, the reactor 205 can facilitate deprotonation of an amino acid (eg, from reservoir 210) with a base (eg, from reservoir 215) such that a carboxylated form of amino acid is produced. In certain embodiments, the reactor 205 may facilitate and / or facilitate the reaction of the reaction product with the reagent from a reservoir located upstream of the reactor 205. For example, the reactor 205 may accelerate the reaction of the carboxylated form of the amino acid with the activator, for example by supplying heat to the reaction. In some such cases, the carboxylate form of the amino acid can be formed upstream of reactor 205 (eg, in or near the mixed region). In other cases, the carboxylate form of the amino acid may be formed in the reactor 205.

In some embodiments, the reactor 205 may be in a heating zone (not shown) or otherwise contact the heat source. For example, the system 200 may include a heating zone (not shown) in which the contents of the fluid stream in the reactor 205 can be heated. The heating zone may include a heat source such as a heater. In general, any suitable heating method can be used to control the temperature of the fluid flow in the reactor. For example, a heating zone produces heat by applying energy, or by applying energy, a liquid bath (eg, a water bath), a resistance heater, a gas convection-based heating element, a microwave heating element, or a chemical reaction. It may include any other suitable device designed to be such. In certain embodiments, the mixed fluid stream does not have to be exposed to a heat source before reaching the reactor. In some embodiments, the mixed fluid flow does not have to be exposed to heat from a heat source between the mixing region and the entrance to the reactor. In some such cases, the temperature of the mixed fluid flow is within about 10 ° C (eg, within about 8 ° C, within about 5 ° C, within about 3 ° C, about) of the temperature of the leading edge at the inlet of the reactor. Within 2 ° C, within about 1 ° C). For example, the temperature of the leading edge is less than about 10 ° C or equal (eg, less than about 8 ° C or equal, less than about 5 ° C or equal, less than about 3 ° C or equal, from the mixing region to the inlet of the reactor. It may vary below about 2 ° C or equal and less than about 1 ° C or equal).

In some embodiments, the system 200 may include two or more reactors. For example, as shown in FIG. 2B, system 200 may include reactor 205 upstream of reactor 235. In certain embodiments, the reactor 205 may be free of a plurality of amino acids immobilized on a solid support and / or a plurality of immobilized peptides. In some such cases, the formation of one or more amino acid residues may not occur within the reactor 205. In some cases, the reagent or reaction product thereof may undergo one or more reactions within the reactor 205. For example, amino acids and bases in the mixed fluid stream may react to produce deprotonated amino acids (eg, amino acids in carboxylate form) in the reactor 205 and / or amino acids in the mixed fluid stream (eg, amino acids in the mixed fluid stream). For example, the carboxylate form of the amino acid) and the activator may react to produce the activated amino acid in the reactor 205. In some embodiments, the formation of one or more amino acid residues may occur within the reactor 235. In some such embodiments, the reactor 235 may contain peptides and / or amino acids immobilized on a solid support. For example, as shown in FIG. 2B, peptide 240 may be immobilized on a solid support 245. The solid support 245 may be contained within the reactor 235.

In some embodiments, the reactor 205 and the reactor 235 may be in direct fluid communication (eg, adjacent, direct connection) as shown in FIG. 2B. Direct fluid communication between reactors may be advantageous. For example, direct fluid communication between the reactor 205 and the reactor 235 may facilitate and / or facilitate the chemical reaction within the reactor 205 just before the fluid flow is exposed to the reactor 235. good. In embodiments where facilitation and / or facilitation involves heating the fluid flow, heating the fluid flow in the reactor 205 immediately prior to exposure to the immobilized peptide in the reactor 235 (to the immobilized peptide, the content of the flow). Pyrolysis of one or more reagents in the stream (eg, amino acids added to the peptide and / or deprotecting reagents), as opposed to heating the stream long before transporting the material. Can be minimized.

In some cases, the reactor 205 may be within a short distance from the reactor 235, eg, within about 5 meters, within about 1 meter, within about 50 cm, or within about 10 cm.

A single reservoir is illustrated in FIGS. 2A and 2B for brevity, but in FIGS. 2A and 2B where a single reservoir is illustrated, multiple reservoirs (eg, respectively) instead of a single reservoir are shown. It should be understood that can use different types of amino acids, different types of activators, different types of bases, different types of additives, etc.). Although the first and second fluid streams are described as containing amino acids and bases, respectively, it should be understood that the first and second fluid streams may contain any suitable activation reagent. Is. For example, the first and second fluid streams described herein may contain activators and bases, respectively.

In some embodiments, the method for solid phase peptide synthesis is to start flowing a first stream comprising a first activation reagent (eg, an amino acid, an activator), a second activation reagent (eg, an activator). For example, starting a second stream containing a base) and merging the first and second fluid streams in a mixed region, including forming a mixed fluid stream with a leading edge. May be good. The mixed fluid flow may flow into the reactor. In some embodiments, merging may include aligning and / or combining leading edges of two or more fluid streams to form a single mixed fluid stream. For example, with reference back to FIGS. 2A and 2B, the flow begins with a reagent reservoir (eg, 100, 210) and is a first fluid flow containing a first activating reagent (eg, an amino acid, an activator). (Eg 130, 250) may be formed, the flow starting from another reagent reservoir (eg 105, 215) and a second fluid flow containing a second activation reagent (eg base). (For example, 135, 255) may be formed. The flow of the first and second flows starts the fluid flow from the reservoir so that the leading edge of the first fluid flow meets the leading edge of the second fluid flow in the mixing region (eg 140, 270). It may be controlled from time to time. In certain embodiments, the leading edge of the first fluid flow and / or the leading edge of the second fluid flow may reach the mixing region within about 10 ms of each other (eg, substantially simultaneously). In some such cases, the leading edge of the first fluid flow and / or the leading edge of the second fluid flow is in the mixed region (eg, 140, 270) prior to the confluence of the flows occurring in the mixed region. It does not flow individually (ie, unmerged) downstream.

In some embodiments, when the leading edges of the first and second flows meet each other within about 10 ms in a mixed region (eg, 140, 270), or otherwise reach, the two or the like. The molar ratio of more activating reagents (eg, amino acids to bases, bases to activators) is the desired ratio for amino acid activation, and / or the amino acids and / or immobilized peptides in the reactor. It may be substantially the same as the desired ratio (eg, during coupling) to prevent side reactions. In some such cases, the molar ratio of the first activator (eg, amino acid, activator) to the second activator (eg, base) in the mixed fluid stream is two or more. More Activating Reagents A mixed fluid flow in the mixed region leading to the introduction of the mixed fluid flow into the reactor from the leading edge and / or slightly offset (eg, less than about 10 ms) leading edge of the fluid flow. It does not change significantly compared to when it was formed (eg, 10% or less, 5% or less). In some such embodiments, a first activating reagent and a second activating reagent (eg, at least one of amino acids and bases, at least one of activators and bases) are added to the reactor. When first reached, the molar ratio of the first activating reagent (eg, amino acid, activator) to the second activating reagent (eg, base) in the mixed fluid stream in the mixed region is the reactor. It is within 10% (for example, 5%) of the molar ratio of the first activation reagent and the second activation reagent in. For example, in some embodiments, when the leading edge of the mixed fluid enters the reactor, a first activation reagent (eg, amino acid, activator) and a second activation at the leading edge of the mixed fluid. The molar ratio with the reagent (eg, base) is within 10% (eg, 5%) of the molar ratio of the first activating reagent and the second activating reagent of the mixed fluid in the mixing region. In some embodiments, the molar ratio at the entrance to the reactor is at least about 10 ms, at least about 50 ms, at least about 100 ms, or at least about 1 second after the time the leading edge enters the reactor. , May vary by less than about 10% (eg, 5%).

In some embodiments, the molar ratio of the first activator (eg, amino acid, activator) to the second activator (eg, base) in the mixed fluid stream in the mixed region is 2. Seed or more activator fluid flow leading edge and / or slightly displaced (eg, less than about 10 ms) leading edge to a subsequent point in time (eg, at least about 10 ms, at least about 50 ms) No significant change (eg, 10% or less, 5% or less) compared to when a mixed fluid flow was formed in the mixed region (at least about 100 ms, or at least about 1 second later). For example, when a mixed fluid flow is formed in the mixed region from the leading edge, the molar ratio of the first activating reagent to the second activator in the mixed region is at least after the mixed fluid flow is formed. Within 10% (eg, 5%, 2%, 1%, 0.5%) of the molar ratio in the mixed region after about 10 ms (eg, at least about 50 ms, at least about 100 ms, or at least about 1 second). You may.

In certain embodiments, the first or second fluid stream may comprise another activation reagent. For example, the second fluid stream may contain a base and an activator. In some such embodiments, the mixed fluid stream may contain activated amino acids due to the activation of amino acids by bases and activators. In some such cases, the mixed fluid stream may be free of excess base and / or activator.

In some embodiments, the first or second fluid stream may not include a separate activation reagent. In some such embodiments, the method for solid phase peptide synthesis is to flow a first stream containing an amino acid, a second stream containing a base, a third stream comprising an activator. It may include flowing a stream and merging the fluid streams in a mixed region to form a mixed fluid stream. The mixed fluid flow may flow into the reactor. In some such embodiments, merging aligns and combines three or more fluid flow leading edges (eg, first, second, and third fluid flow leading edges). / Or combined may include forming a single mixed fluid stream with a leading edge. For example, referring to FIGS. 2A and 2B, the stream may start with a reservoir containing amino acids (eg, 100, 210) and form a first stream (eg, 130, 250), where the stream is base. A second stream (eg, 135, 255) may be formed starting from another reservoir containing (eg, 105, 215), the stream starting from a different reservoir (eg, 110, 220). A third flow (eg, 145, 260) may be formed. In some embodiments, when the anterior edges of the third fluid flow meet or reach each other within about 10 ms of each other in a mixed region (eg, 140, 270), two or more of the activation reagents. More molar ratios (eg, amino acids and bases, and / or bases and activators) are the desired ratios for amino acid activation, and / or amino acids and / or immobilized peptides in the reactor. It may be substantially the same as the desired ratio (eg, during coupling) to prevent side reactions. In some such cases, two or more molar ratios in the mixed fluid stream (eg, first activation reagent and second activation reagent, first activation reagent and third The activation reagent, and / or the second and third activation reagents, were offset by the leading edge and / or slightly of the fluid flow of three or more activation reagents (less than about 10 ms). There is no significant change (eg, 10% or less, 5% or less) compared to when a mixed fluid flow is formed in the mixed region leading from the (shifted) front edge to the introduction of the mixed fluid flow into the reagent. In some such embodiments, at least one of the first, second, and third activation reagents (eg, at least two of them) is first in the reactor. When the first activation reagent (eg, amino acid) and the second activation reagent (eg, base), the first activation reagent (eg, amino acid) in the mixed fluid flow in the mixed region The molar ratio of the third activating reagent (eg, activator) and / or the second activating reagent (eg, base) to the third activating reagent (eg, activator) is the reactor. Within 10% (eg, 5%) of the molar ratio (s). For example, in some embodiments, when the leading edge of the mixed fluid enters the reactor, the molar ratio (s) at the leading edge of the mixed fluid is the molar ratio (s) in the mixed region. Within 10% (eg, 5%). In some embodiments, the molar ratio (s) at the entrance to the reactor is at least about 10 ms, at least about 50 ms, at least about 100 ms, or at least about 1 from the time the leading edge enters the reactor. It may change by less than about 10% (eg, 5%) until after seconds.

In some embodiments, with two or more molar ratios (eg, a first activator and a second activator, a first activator) in a mixed fluid stream in a mixed region. The third activator and / or the second activator and the third activator) were slightly offset (and / or slightly offset) from the leading edge of the fluid flow of the three or more activators. For example, when a mixed fluid flow is formed in a mixed region from the leading edge (offset by less than about 10 ms) to a point in time thereafter (eg, at least about 10 ms, at least about 50 ms, at least about 100 ms, or at least about 1 second later). Does not change significantly (for example, 10% or less, 5% or less). For example, when a mixed fluid flow is formed in the mixed region from the leading edge, the molar ratio in the mixed region is at least about 10 ms (eg, at least about 50 ms, at least about 100 ms, or at least) after the mixed fluid flow is formed. It may be within 10% (for example, 5%, 2%, 1%, 0.5%) of the molar ratio in the mixed region after about 1 second).

In certain embodiments, the method for solid phase peptide synthesis is to apply a fourth fluid stream containing one or more additives in the mixed region (eg, junction), first, second, and. Combining with one or more of the third fluid streams may include forming the mixed fluid streams described above. For example, with reference to FIGS. 2A and 2B, the flow begins with an optional reservoir containing one or more additives (eg, 120, 225) and a fourth fluid flow (eg, 155, 265). It may be formed and reach a mixed region (eg 140, 270). In some cases, the leading edge of the fourth fluid flow may be merged with the leading edge of the first, second, and third fluid streams at the mixing region (eg, the junction). In such cases, the molar ratio of activation reagents (eg, amino acids, activators, bases) may not be substantially altered by the addition of a fourth fluid stream as described above (5%). Less than).

In some embodiments, during the merging process, two or more fluid flows (eg, first and second fluid flows; first, second, and third fluid flows; first, first. The leading edges of the second, third, and fourth fluid streams, all) may reach the mixing region within a relatively short period of time with each other. For example, two or more (eg, three or more, four or more) fluid streams are within about 25 ms, within about 22 ms, within about 20 ms, within about 18 ms, about each other. The mixed region may be reached within 15 ms, within about 10 ms, within about 9 ms, within about 8 ms, within about 7 ms, within about 6 ms, within about 5 ms, or within about 4 ms. In some embodiments, the time it takes for the fluid flow to reach the mixing region is a UV-vis detector positioned on each activation reagent fluid flow adjacent to the mixing region, as well as downstream of the mixing region. It may be determined using an adjacently positioned UV-vis detector. As the fluid begins to flow, the detector makes continuous measurements over time until a signal indicating the mixed fluid is measured by a UV-vis detector located downstream of the mixing region. The absorbance vs. time curves created from the UV-vis measurements are superimposed on each other. The time difference between fluid detections is used to determine the time it takes for two or more fluids to reach the mixed region. It should be noted that each UV-vis detector is set to an appropriate wavelength for measuring the associated fluid flow.

In certain embodiments, the reactor and / or immobilized peptide may be exposed to the mixed fluid stream for a relatively short period of time after the fluid streams have merged. For example, in certain embodiments, the peptides immobilized on the reactor and / or solid support are such that a mixed fluid stream is formed after the fluid stream merges (eg, first and second). Fluid flow; 1st, 2nd, and 3rd fluid flow; 1st, 2nd, 3rd, and 4th fluid flow), within about 30 seconds (or within about 15 seconds, within about 10 seconds, about) It may be exposed to the mixed fluid for 5 seconds or less, about 3 seconds or less, about 2 seconds or less, about 1 second or less, about 0.1 seconds or less, or about 0.01 seconds or less). In some embodiments, the residence time of the mixed fluid flow from the mixing region (eg, the junction) to the reactor is at least about 0.1 seconds and less than about 30 seconds, at least about 0.1 seconds and about 25 seconds. Less than, at least about 0.1 seconds and less than about 20 seconds, at least about 0.1 seconds and less than about 15 seconds, at least about 0.1 seconds and less than about 10 seconds, at least about 1 second and less than about 30 seconds, at least about about 1 second and less than about 25 seconds, or at least about 1 second and less than about 15 seconds. As used herein, residence time refers to the total time required for a fluid flow to transition from the mixing region to the inlet of the reactor.

In certain embodiments, at least a portion of the mixed fluid flow is positioned between the mixing region (eg, the junction) and the reactor after the fluid flows have merged but before being introduced into the reactor. It may flow into the mixed mixer. The mixer can facilitate the formation of a homogeneous fluid flow by facilitating active and / or passive mixing. In general, any suitable mixer may be used and one of ordinary skill in the art will be familiar with active and passive mixers.

In some embodiments, the molar ratio between the two activation reagents in the mixed region (eg, amino acids to bases, bases to activators, amino acids to activators) and similar molar ratios in the reactor. And / or the molar ratio between the two activation reagents in the mixed region at the first time and similar molars at the later time point (eg, the second time). The difference from the ratio is less than about 10% or equal, less than about 8% or equal, less than about 6% or equal, less than about 5% or equal, less than about 4% or equal, less than about 2%. Or equal to, less than about 1% or equal to it, less than about 10% or equal to it, less than about 10% or equal to it, or less than about 0.5% or equal to it. For example, in some embodiments, the molar ratios between the two activators (eg, amino acids to bases, bases to activators, amino acids to activators) deviate from the leading edge and / or slightly. In the mixed region leading to the introduction of the mixed fluid flow into the reactor from the leading edge (eg, offset by less than about 10 ms) and / or from the first time point in the mixed region to a later point in time in the mixed region. From the time of formation, it changes by 10% or less (for example, 8% or less, 6% or less, 5% or less, 4% or less, 2% or less, 1% or less, 0.5% or less).

In some embodiments, the molar ratio at the entrance to the reactor is at least about 10 ms, at least about 15 ms, at least about 25 ms, at least about 50 ms, at least about 75 ms from the time the leading edge enters the reactor. Less than about 10% or equal, less than about 8% or equal, less than about 6% or equal, less than about 5% or equal, less than about 4%, at least about 100 ms, or at least about 1 second later. Or equally, less than about 2% or equal to it, less than about 1% or equal to it, less than about 10% or equal to it, less than about 10% or equal to it, or less than about 0.5% or equal to it. good.

として計算され得る。 When calculating the percentage of the difference between two values (unless otherwise specified herein), the percentage calculation is basically done using values that are larger in magnitude. For example, if the first value is V ₁ and the second value is V ₂ (greater than V ₁ ), then the percentage difference between V ₁ and V ₂ (V _{% Diff} ) is.

Can be calculated as.

The first and second values can be said to be within X% of each other if V _{% Diff} is less than X%. The first and second values can be said to differ by at least X% if V _{% Diff} is X% or greater.

In some embodiments, the molar ratio of one activation reagent to another (eg, base to activator, amino acid to base, amino acid to activator) is at least about 1: 0.7 and It may be less than about 2: 1. For example, the molar ratio of the first activation reagent to the second activation reagent (eg, amino acids to bases, bases to activators, activators to bases) is at least about 1: 0.7 and about 2. Less than 1: (eg, at least about 1: 0.8 and less than about 2: 1, at least about 1: 0.9 and less than about 2: 1, at least about 1: 1 and less than about 2: 1, at least about 1: 0.7 and less than about 1.9: 1, at least about 1: 0.7 and less than about 1.8: 1, at least about 1: 0.7 and less than about 1.6: 1, at least about 1: 0. 7 and less than about 1.5: 1, at least about 1: 0.7 and less than about 1.4: 1, between about 1: 0.7 and about 1.2: 1, about 1: 0.7 to about It may be between 1: 1). In some embodiments, the molar ratio of the first activating reagent (eg, activator) to the second activating reagent (eg, base) may be at least 1: 1. In some embodiments, the molar ratio is a UV-vis detector positioned adjacent to the mixing reagent on each activation reagent fluid stream, a UV-vis detector positioned downstream of the mixing region, and a reaction. It may be determined using a UV-vis detector positioned at the entrance of the vessel. When the fluid begins to flow, the detector makes continuous measurements until a signal indicating the mixed flow is measured downstream and adjacent to the mixed region by a positioned UV-vis detector. When the signal indicating the mixed fluid is measured by a UV-vis detector located downstream of the mixing region, the UV-vis measurement is at the wavelength required to determine the concentration of each fluid in the mixed fluid flow. It is performed every 25 ms. The absorbance vs. time curves created from the UV-vis measurements are superimposed on each other. The concentration of each activation reagent at each relevant location is determined from the curve.

In general, the streams may be merged using any suitable technique known to those of skill in the art. In some embodiments, the flow is such that the fluid flows (eg, first, second, third, and / or fourth) flow substantially simultaneously to form a single flow (eg, flow). It may be merged (by merging the channels flowing inside). For example, referring to FIGS. 2A and 2B, fluid flows 130, 135, 145, respectively, using optional flow controllers (eg, pumps) 101, 102, 103, 104, 201, 202, 203, and 204, respectively. Flow rates and arrival times of 1, 155, 250, 255, 260, and 265 may be controlled. At least a portion of the flow controller may be configured to allow merging as described herein. In such cases, the pump may be configured such that the dead volume of the head and the upstream fluid are substantially the same (eg, identical). In some cases, the lengths of 130, 135, 145, 250, 255, or 260 in FIGS. 2A and 2B may be adjusted to compensate for differences in internal volume. The pumps are then started within certain time periods with each other (eg, within about 100 ms, within about 80 ms, within about 60 ms, within about 50 ms, within about 40 ms, within about 25 ms, within about 10 ms, within about 5 ms). Two or more pumps may be connected together in such a manner.

As described herein, methods and systems for amino acid activation may be used in the solid phase peptide synthesis described in more detail below. In general, solid phase peptide synthesis involves repeating an amino acid addition cycle that includes a deprotection reaction, a coupling reaction, and an optional reagent removal (eg, wash) step. In some embodiments, the confluence of activation reagent streams may be used in one or more amino acid addition cycles as described in more detail below for solid phase peptide synthesis. For example, the first fluid stream containing amino acids and the second fluid stream containing bases are combined and mixed within about 30 seconds prior to exposure of the activated amino acids to the peptide immobilized on the solid support. A fluid flow may be formed. In some embodiments where more than one amino acid addition cycle occurs during solid phase peptide synthesis, one or more amino acid addition cycles (eg, first and second amino acid addition cycles) comprise amino acids. It may include merging the first fluid stream with the second fluid stream containing the base to form a mixed fluid stream within about 30 seconds prior to exposing the amino acid to the solid support.

Exemplary amino acid addition cycles and peptide synthesis are described in more detail here. In some embodiments, the process of adding an amino acid residue to an immobilized peptide involves exposing the deprotecting reagent to the immobilized peptide to remove at least a portion of the protecting group from at least a portion of the immobilized peptide. include. The deprotective reagent exposure step can be configured in certain embodiments so that the side chain protecting group is preserved while the N-terminal protecting group is removed. For example, in certain embodiments, the protecting group used to protect the peptide comprises fluorenylmethyloxycarbonyl (Fmoc). In some such embodiments, a deprotective reagent containing piperidine (eg, a piperidine solution) may be exposed to the immobilized peptide so that the Fmoc protecting group is removed from at least a portion of the immobilized peptide. .. In some embodiments, the protecting group used to protect the peptide comprises butyloxycarbonyl (Boc). In some such embodiments, a deprotective reagent containing trifluoroacetic acid may be exposed to the immobilized peptide so that the Boc protecting group is removed from at least a portion of the immobilized peptide. In some cases, a protecting group (eg, tert-butoxycarbonyl, i.e., Boc) may be attached to the N-terminus of the peptide.

In some embodiments, the process of adding an amino acid residue to the immobilized peptide comprises removing at least a portion of the deprotecting reagent. In some embodiments, at least a portion of any reaction by-product (eg, removed protecting group) that may be formed during the deprotection step can be removed. In some cases, the deprotecting reagent (in certain embodiments, a by-product) is a fluid (eg, a by-product) conserving the peptide, solid support, and / or surrounding area, eg, in an optional reservoir 125. , Aqueous or non-aqueous solvents, supercritical fluids, and / or liquids such as the like). In some cases, removal of deprotecting reagents and reaction by-products can improve the performance of subsequent steps (eg, by preventing side reactions). In certain embodiments, the performance of subsequent steps does not depend on the removal of at least a portion (eg, substantially all) of the deprotecting reagent and / or reaction by-product. In some such cases, the removal step is optional. In embodiments where the removal step is optional, the removal step may be reduced (eg, reduced time in the removal step, reduced amount of solvent used in the removal step) and / or excluded. Reducing one or more removal steps can reduce the total cycle time. If the optional removal step is reduced or excluded, subsequent steps during the addition cycle remove at least a portion of the deprotecting reagent and / or reaction product, for example due to fluid flow in the system. It should be understood that it can play a role.

The process of adding an amino acid residue to an immobilized peptide, in certain embodiments, is novel by exposing the activated amino acid to the immobilized peptide and binding at least a portion of the activated amino acid to the immobilized peptide. Includes allowing bound amino acid residues to form. For example, the peptide may be exposed to an activated amino acid that reacts with the deprotected N-terminus of the peptide. In certain embodiments, amino acids can be activated towards reaction with the deprotected peptide by mixing an amino acid-containing stream with an activator stream, as discussed in more detail below. In some cases, the amine group of the activated amino acid may be protected so that the addition of the amino acid results in an immobilized peptide with a protected N-terminus. In some embodiments, the peptide may be exposed to an activating amino acid that reacts with the deprotected side chain of the immobilized peptide.

In some embodiments, the process of adding an amino acid residue to an immobilized peptide comprises removing at least a portion of the activated amino acid that does not bind to the immobilized peptide. In some embodiments, at least some of the reaction by-products that may form during the activated amino acid exposure step may be removed. In some cases, the activated amino acids and by-products are, for example, in a fluid stored in an optional reservoir 125 (eg, a liquid such as an aqueous or non-aqueous solvent, a supercritical fluid, and / or the like). , Peptides, solid supports, and / or may be removed by washing the surrounding area. In some cases, removal of at least a portion of the activated amino acids and reaction by-products can improve the performance of subsequent steps (eg, by preventing side reactions). In certain embodiments, the performance of subsequent steps does not depend on the removal of at least some (eg, substantially all) of the activated amino acids and / or reaction by-products. In some such cases, the removal step is optional. In embodiments where the removal step is optional, the removal step may be reduced (eg, reduced time in the removal step, reduced amount of solvent used in the removal step) and / or excluded. Reducing or excluding one or more removal steps may reduce the overall cycle time. If the optional removal step is reduced or excluded, subsequent steps during the addition cycle remove at least some of the activated amino acids and / or reaction by-products, for example due to fluid flow in the system. It should be understood that it can play a role in

It should be understood that the steps mentioned above are exemplary and that the amino acid addition cycle does not necessarily include all of the steps mentioned above. For example, the amino acid addition cycle may not include a deprotecting reagent removal step and / or an activated amino acid removal step. In general, the amino acid addition cycle comprises any sequence of steps that results in the addition of amino acid residues to the peptide.

In certain embodiments, the addition of amino acids during the amino acid addition step results in a peptide incorporating a single additional amino acid residue (ie, a single amino acid residue is given after the addition step). Can be added to the immobilized peptide such that the peptide contains a single additional amino acid residue). In some such embodiments, subsequent amino acid addition steps can be used to construct the peptide by individually adding amino acid residues until the desired peptide is synthesized. In some embodiments, more than one amino acid residue (eg, in the form of a peptide) may be added to the peptide immobilized on the solid support (ie, multiple amino acid residues). Peptides containing the above can be added to a given immobilized peptide). Addition of the peptide to the immobilized peptide can be achieved through processes known to those of skill in the art (eg, fragment condensation, chemical ligation). That is, during the amino acid addition step, addition of an amino acid to the immobilized peptide adds a single amino acid residue to the immobilized peptide or adds multiple amino acid residues (eg, as a peptide) to the immobilized peptide. Can include that.

In certain embodiments, the first amino acid addition step (and / or subsequent amino acid addition step) may add amino acids in relatively high yields. For example, in certain embodiments, amino acids are added such that amino acid residues are added to at least about 99%, at least about 99.9%, at least about 99.99%, or substantially 100% of the immobilized peptide. Includes exposure to the immobilized peptide. In certain embodiments, the amino acid is immobilized so that the amino acid residue is added to at least about 99%, at least about 99.9%, at least about 99.99%, or substantially 100% of the immobilized peptide. A second (in some embodiments, a third, fourth, fifth, and / or subsequent) amino acid addition cycle may include exposure to the peptide. In certain embodiments, the use and system of the processes of the invention relatively rapidly add more than one amino acid to the immobilized peptide while maintaining high reaction yields (discussed above). Alternatively, it may be possible to do so (including within any of the time ranges disclosed elsewhere in this specification).

In certain embodiments, one or more amino acid addition steps can be performed with little or no double integration (ie, the desired amino acid (eg, a single addition step) during a single addition step. Add multiple replicas of amino acid residues or peptides)). For example, in certain embodiments, multiple replicas of the desired amino acid are fixed during the first (and / or second, third, fourth, fifth, and / or subsequent) amino acid addition steps. Less than about 1% (or less than about 0.1%, less than about 0.01%, less than about 0.001%, less than about 0.0001%, about 0. Bind to less than 0001%, or substantially none).

In some embodiments, multiple amino acid addition cycles can be performed. By performing multiple amino acid addition cycles, more than one single amino acid residue (or more than one peptide, and / or at least one single amino acid residue, and at least One peptide) can be obtained. In certain embodiments, the process for adding more than one amino acid to the immobilized peptide is to perform a first amino acid addition cycle to add the first amino acid, and to add a second amino acid. It may include performing a cycle to add a second amino acid. In certain embodiments, third, fourth, fifth, and subsequent amino acid addition cycles may be performed to produce an immobilized peptide of any desired length. In some embodiments, at least about 10 amino acid addition cycles, at least about 50 amino acid addition cycles, or at least about 100 amino acid addition cycles are performed, resulting in at least about 10 amino acid residues on the immobilized peptide. , At least about 50 amino acid residues, or at least about 100 amino acid residues are added. In certain such embodiments, a relatively high percentage of amino acid addition cycles (eg, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least of such amino acid addition cycles. About 99%) can be done in high yields (eg, at least about 99%, at least about 99.9%, at least about 99.99%, or substantially 100%). In some such embodiments, a relatively high percentage of amino acid addition cycles (eg, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about such amino acid addition cycles. About 99%) can be done quickly, for example, within any of the time ranges specified above or elsewhere herein. In some such embodiments, a relatively high percentage of amino acid addition cycles (eg, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least of such amino acid addition cycles. Approx. ..

In some embodiments, solid phase peptide synthesis may include heating the flow before reaching the reactor but within a short time to reach the reactor. By supplying a heating stream to the reactor, the dynamics of the processes initiated within the reactor may be altered. For example, the reaction rate and / or diffusion rate of the amino acid addition process may be varied by exposing the immobilized peptide, solid support, or other synthetic component to a heated stream. For example, exposure of the peptide to a heated stream containing activated amino acids may increase the rate at which the amino acids are added to the peptide. In some embodiments, auxiliary heat (ie, from one or more preheated streams) by heating the stream before it arrives at the reactor but within a short time to reach the reactor. No heat) may be substantially reduced or eliminated from the need to supply the reactor. In some cases, almost or substantially all of the heat delivered to the reactor comes from the preheating stream. For example, in some embodiments, the percentage of thermal energy used to heat the reactor from the preheating stream (s) is greater than or equal to about 50% and greater than about 60%. Or equal to it, greater than or equal to about 70%, greater than or equal to about 80%, greater than or equal to about 90%, greater than or equal to about 95% or equal to, or greater than about 99%. It may be large or equal. In some such embodiments, heating the system in this way provides a reactor, immobilized peptide, solid support, activated amino acid, deprotecting reagent, washing fluid, and / or other synthetic components. , The time required to heat to the desired reaction temperature can be reduced.

In some embodiments, the process for adding an amino acid residue to a peptide is such that the temperature of the activating amino acid is at least about 1 ° C (or at least about 2 ° C, at least about 5 ° C, at least about 10 ° C, at least about 25 ° C. A stream containing the activating amino acid is heated so that the temperature rises to at least about 50 ° C. and / or less than or equal to about 100 ° C. and / or less than or equal to about 75 ° C., and then the heated amino acid. May include exposure to the immobilized peptide. In certain embodiments, a stream containing any other component (eg, a cleaning agent, a deprotecting agent, or any other component) has a flow content that has a temperature of at least about 1 ° C (or at least about 2). Heat to rise at ° C, at least about 5 ° C, at least about 10 ° C, at least about 25 ° C, at least about 50 ° C, and / or less than or equal to about 100 ° C and / or less than or equal to about 75 ° C. After that, the contents of the stream may be exposed to the immobilized peptide. In some cases, the heating step (eg, heating of the activated amino acid and / or any other component in the stream transported to the immobilized peptide) is the content of the stream (eg, heated activated amino acid). ) Within about 30 seconds (or within about 15 seconds, within about 10 seconds, within about 5 seconds, within about 3 seconds, within about 2 seconds, within about 1 second, about 0.1 seconds after exposure to the immobilized peptide) Within, or within about 0.01 seconds). In some such embodiments, such heating may be achieved by heating the upstream position of the immobilized peptide. In some such embodiments, heating of the amino acid begins at least about 0.1 seconds, at least about 1 second, at least about 5 seconds, or at least about 10 seconds before exposing the amino acid to the immobilized peptide. do. In certain embodiments, the amino acid is at least about 1 ° C. (at least about 0.1 seconds, at least about 1 second, at least about 5 seconds, or at least about 10 seconds before exposing the amino acid to the immobilized peptide). Or at least about 2 ° C, at least about 5 ° C, at least about 10 ° C, at least about 25 ° C, at least about 50 ° C, and / or less than or equal to about 100 ° C and / or less than or equal to about 75 ° C). To.

In some embodiments, heating of the amino acid and confluence of the amino acid with the base and / or activator are both performed before and within a relatively short period of time after the amino acid comes into contact with the immobilized peptide. Can be done. Amino acid heating may be performed before, during, and / or after merging the streams.

In general, any protecting group known to those of skill in the art can be used. Non-limiting examples of protecting groups (eg, n-terminal protecting groups) include fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, allyloxycarbonyl (alloc), carboxybenzyl, and photodissociative protecting groups. Is done. In certain embodiments, the immobilized peptide comprises a fluorenylmethyloxycarbonyl protecting group. In some embodiments, the immobilized peptide comprises a tert-butyloxycarbonyl protecting group.

As described herein, the base may be used to activate or complete the activation of the amino acid prior to exposure to the immobilized peptide. Any suitable base may be used. In certain embodiments, the base is a Lewis base. In some embodiments, the base is triisopropylamine, N, N-diisopropylethylamine, which is non-reactive to the protected peptide or slowly reacts with the protected peptide to remove the protecting group. Certain tertiary amines, or non-nucleophilic bases such as colisine. In general, the base may have sufficient pKa to allow deprotonation of amino acids to carboxylic acids.

As described elsewhere, activators may be used to form bonds with the C-terminus of amino acids to facilitate coupling reactions and the formation of amide bonds. The activator may be used to form the activating amino acid before exposing the amino acid to the immobilized peptide. Any suitable activator may be used. In some embodiments, the activator is selected from the group consisting of carbodiimides, guanidinium salts, phosphonium salts, and uronium salts. Activators include, in some embodiments, carbodiimides such as N, N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and the like. .. In certain embodiments, the activator is a uronium activator such as O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU); 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); 1-[(1- (cyano-2-ethoxy-2) -Oxoethylideneaminooxy) dimethylaminomorpholino)] uronium hexafluorophosphate (COMU); and the like. In certain embodiments, the activator comprises a phosphonium activator, such as (benzotriazole-1-yloxy) tripyrolidinophosphonium hexafluorophosphate (PyBOP).

As described elsewhere, the peptide may be immobilized on a solid support. In general, any solid support may be used with any of the addition cycles described herein. Non-limiting examples of solid support materials include polystyrene (in resin forms such as microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin), glass, polysaccharides (eg cellulose, agarose). , Polyacrylamide resin, polyethylene glycol, or copolymer resin (including, for example, polyethylene glycol, polystyrene, etc.).

The solid support may have any suitable shape factor. For example, the solid support can be in the form of beads, particles, fibers, or any other suitable shape factor.

In some embodiments, the solid support may be porous. For example, in some embodiments, a macroporous material (eg, macroporous polystyrene resin), a mesoporous material, and / or a microporous material (eg, microporous polystyrene resin) is used as the solid support. May be good. The terms "macroporous", "mesoporous", and "microporous" are known to those of skill in the art when used in connection with solid supports for peptide synthesis and are herein known. , The International Union of Pure and Applied Chemistry (IUPAC) Compendium of Chemical Terminology, 2.3.2 edition, August 19, 2012 (unofficially known as "Gold Book") and inconsistent with their description. Used in the absence. Generally, microporous materials include those having pores with a cross-sectional diameter of less than about 2 nanometers. Mesoporous materials include those having pores with a cross-sectional diameter of about 2 nanometers to about 50 nanometers. Macroporous materials include those having pores larger than about 50 nanometers and having a cross-sectional diameter as large as about 1 micrometer.

As used herein, the term "peptide" has the usual meaning in the art and is a covalent bond from one carbonyl carbon to another nitrogen atom in the presence of conventional water loss. It may refer to an amide derived from two or more aminocarboxylic acid molecules (same or different) by formation. "Amino acid residue" also has its usual meaning in the art and is a composition of amino acids (as a single amino acid or as part of a peptide) after being combined with a peptide, another amino acid, or an amino acid residue. ). Generally, when an amino acid is combined with another amino acid or amino acid residue, water is removed and the rest of the amino acid is called an amino acid residue. The term "amino acid" also has its usual meaning in the art and may include proteoplastic and non-proteinogenic amino acids. In some embodiments, the amino acid may be in carboxylate form. In some embodiments, the amino acid may be in the form of a carboxylic acid.

The term "protecting group" as used herein is given its usual meaning in the art. The protecting group adhered to the reactive group (ie, the protected group) within the molecule (eg, peptide) to prevent or otherwise otherwise block the group protected by the protecting group from reacting. Or it contains chemical moieties that are configured to adhere. Protection may be provided by attaching a protecting group to the molecule. Deprotection may occur when the protecting group is removed from the molecule, for example by a chemical conversion that removes the protecting group.

The following examples are intended to exemplify certain embodiments of the invention and do not exemplify the entire scope of the invention.

(Example 1)
The device shown in FIG. 3 includes three pumps connected downstream of the fluid manifold where the fluid flows meet at one point. One of the three pumps was connected upstream of the manifold for selecting amino acids from one of many reservoirs. The second pump is connected upstream of the manifold for selecting an activator from one of many reservoirs. A third pump was connected upstream of a reservoir containing a base, or a manifold for selecting a base from one of many reservoirs.

To perform the coupling step described in the subsequent examples, the valves were switched to select the desired activator, amino acid, and base. The pump is then operated so that the leading edges of the three fluid streams meet at the confluence at the same time. This pumping cycle continued until the operator or software attempted to stop the coupling and replace the valve with a wash solvent. The pump was then operated to align the trailing edges of the fluid flow.
(Example 2)

This example describes peptide synthesis using the merging technique described in Example 1 and peptide synthesis using conventional methods.

Briefly, the sequence shown in FIG. 3B, EETI-II, was synthesized using the activation method of Example 1. As a control, the same peptide was synthesized using a method in which the tips of the fluid streams did not merge at the same time. Both peptides were synthesized using HBTU at 70 ° C. with a total flow rate of 20 mL / min using standard synthetic parameters.

In control synthesis, the ratio of activators in the fluid stream was the desired ratio (s) since no substantially simultaneous confluence occurred, LC- of the crude peptide shown in FIG. 3B (previous). Significant cuts were observed in the MS locus. The cut was a slug of unreacted activator introduced into the reactor. As shown in FIG. 3C, when the activating reagent reaches the mixed region before another activating reagent, the molar ratio of activating reagent changes in the fluid flow. After matching the concentration profile of the fluid flow using the method described in Example 1, the crude peptide has no cleavage and is even more easily purified as shown in FIG. 3B (later). rice field.
(Example 3)

This example describes the effect of pump typing on substantially simultaneous confluence of fluid flows to activate amino acids. Pump timing inconsistencies resulted in side reactions.

Briefly, peptide ACPs were synthesized by varying the flow rate using a solid phase peptide synthesizer with inconsistent pump timing, so the activation reagent did not reach the junction at the same time. Due to pump typing inconsistencies, reagent flow differences become more exaggerated at high flow rates. This was embodied by the presence of the cleavage product, (TMG) -AAIDYING, indicated by the arrow in FIG. 4A. The cleavage was the result of coupling between the unmixed activator and the immobilized peptide. As the flow rate increased, the proportion of cleavage products in the resulting LC-MS chromatogram increased, but the deletion by-products remained relatively constant.
(Example 4)

This example describes peptide synthesis using the confluence technique described in Example 1.

Amide bond formation reactions are widely used in the synthesis of small molecules, peptides, and proteins for therapeutic agents. Of the 128 recently investigated small molecule drug candidates, 65% required the formation of amides. In addition to small molecules, peptides containing GLP-1 agonists for the treatment of diabetes require the formation of up to 40 amide bonds. Personalized peptide vaccines, which are at the forefront of cancer treatment, require custom synthesis for each patient. However, the research, development, and production of these peptides is limited by the rate of synthesis and typically takes minutes to hours for each amino acid addition and deprotection cycle. In this example, we report a fully automated flow chemistry technique for solid phase polypeptide synthesis by forming an amide bond in 7 seconds and describe a complete cycle time in 40 seconds. The quality of the crude peptide and the isolated yield were comparable to standard batch solid phase peptide synthesis. At full operation, the machine was able to synthesize 25,000 30-mer individual peptides per year, weighing 25 kilograms in total.

Peptides and proteins are important in the search for new therapeutic agents. Research on the underlying peptides and proteins requires the design of new functional variants and the rapid repetition of these designs. Biological expression of peptides can be accelerated and scaled-the ribosome synthesizes peptides at a rate of peptide bond binding of 15 per second-but except for 20 naturally occurring amino acids. It will be difficult. On the other hand, despite the increased number of monomers, chemical peptide synthesis remains relatively slow. This example describes Automated Flow Peptide Synthesis (AFPS), a method that has the flexibility of chemical synthesis to approach the rate of ribosomes. AFPS reduces the amide bond formation step to 7 seconds, reduces the entire cycle for each amino acid addition to 40 seconds, and maintains a high level of control of chemical properties. UV monitoring and disposable reactors enable yield quantification and fast automated switching.

The automated flow peptide synthesizer consists of the five modules shown in FIGS. 5A-5B. During the coupling reaction, the machine withdraws the reagent from the storage module and then mixes the desired amino acid with an amine base (diisopropylethylamine, DIEA) and an activator (eg HATU or PyAOP) in the mixing module. This mixture is an activation module, an electrically heated plug-flow reactor that flows through a reactor that is rapidly heated to 90 ° C. Within 2 seconds of activation, the activated amino acids flow through the coupling module, packed bed of peptide synthetic resin, where the formation of amide bonds is completed within 7 seconds. The resin is housed in a 6 mL disposable syringe cartridge for easy removal. AFPS monitors Fmoc removal at each cycle by recording the absorbance of the reactor effluent as a function of time. The Fmoc-removed absorbance chromatogram makes it possible to infer deprotection efficiency, coupling yield, and the speed of material flux through the peptidyl resin, which allows identification of peptide aggregation on the resin.

AFPS was first validated by synthesizing the test peptide _ALFALFA and a fragment of the acyl carrier protein (ACP 65-74), as shown in FIG. 5D. These peptides were synthesized in high yields with low levels of by-products. Comparative studies were then performed between AFPS, batch synthesis, and longer peptides produced by reliable custom peptide vendors, as shown in FIGS. 6A-6B. Compared to standard batch methods, peptide synthesis using fast continuous flow activation at high temperatures allowed the synthesis of equivalent or higher quality long polypeptides in less time. In addition, as shown in FIG. 6C, in-process UV monitoring provided information on the synthesis yield of each step. The steady decrease in peak area observed for the insulin B chain was obtained from a chain arrest side reaction. These by-products appeared as a series of impurities around the main peak in the LC-MS chromatogram.

The epimerization of Cys and His by high temperature flow activation was then evaluated. Upon activation, Cys and His can lose their stereochemical properties at the ^Cα position. This problem complicates batch synthesis techniques, especially at high temperatures, because activation, coupling, and decomposition all occur simultaneously in the same tank. In a batch microwave synthesizer, the undesired D-Cys product 16 by coupling Fmoc-L-Cys (Trt) at 90 ° C. for 1.5 minutes under microwave irradiation with HBTU and DIEA. It was found that 0.7% was formed. In contrast, in continuous flow, it was found that the length of time in the heating zone of the system shown in FIG. 7A makes the activation process controllable. To explore this, we used two model peptides, FHL and GCF, whose diastereomers can be separated and quantified by LC-MS. By increasing the flow rate and thus reducing the residence time at the temperature of activated Fmoc-Cys (Trt) and Fmoc-His (Boc), diastereomeric formation was performed at 0. Limited to 5% and 3% in GCF. This level of diastereomer formation is consistent with an optimized room temperature batch synthesis protocol.

The methods described in this example offer a number of advantages over manual flow synthesis, heat accelerated batch synthesis, and other continuous flow peptide synthesis methods. First, the automation of the entire process of mixing and heating, mixing, and activating amino acids in a mix-and-match format adjusts the chemical properties on a residue-by-residue basis. Brings infinite possibilities. Second, in-line mixing of these reagents by precise pump and valve operation allows control of stoichiometry, residence time, and amino acid epimerization, resulting in very high synthetic reproducibility.

FIG. 5A shows a photograph of an automated flow solid phase synthesizer, highlighting different system modules and process flow charts. Amino acids, activators, and DIEA are merged together by three HPLC pumps. A series of multi-position valves control the selection of amino acids and activators. Amino acid activation is triggered by flow through one of several heated channels, which is determined by the location of the column switching valve. The activated amino acid then flows over a resin floor containing 200 mg of peptidyl resin contained in a 6 mL frit polypropylene syringe covered with a heated jacket. The waste effluent passes through a UV-visible spectrometer and is then discarded. FIG. 5B shows a cycle diagram showing the duration of each step, the solution composition in each step after mixing, and the total volume of reagents used in each step. FIG. 5C shows LC-MS data for a crude product of acyl carrier protein (65-74) synthesis using Method B, synthesized with an isolated yield of 44%. In this synthesis, 200 mg of starting peptidyl resin resulted in 314 mg of dry resin. Throughout this study, the yield of isolated crude peptides is based on the nominal loading of the resin. FIG. 5D shows an example of UV absorbance data for one coupling and deprotection cycle.

FIG. 6A shows LC-MS data of growth hormone-releasing hormone synthesized via different methods. Growth hormone-releasing hormone was synthesized by (i) Method A in 40 minutes with a 58% isolation yield and (ii) with a 60% isolation yield in 30 hours using a manual batch technique. Compared. The peptide was also purchased from two vendors (iii, iv) with a lead time of 6 weeks. Cleavage of 200 mg of each of these peptidyl resins resulted in 76 mg and 90 mg, equivalent amounts to automated and manual synthesis. FIG. 6B shows LC-MS data for insulin B chains synthesized using different methods. Insulin B chains were synthesized using Method B in 20 minutes with 53% isolated yields and compared to 30 hours and 45% yields by manual batch technique. FIG. 6C shows a plot of Fmoc deprotected UV data for each cycle of synthesis for GHRH and insulin B chains. The peak area, full width at half maximum, and maximum peak value are plotted as a function of the number of couplings. Liquid chromatography and ESI-MS were performed on an Agilent 1260 Infinity LC coupled to a 6520 QTOF mass spectrometer. Each sample was injected into a Zorbax 300SB-C3 column pre-equilibrium with 5% aqueous acetonitrile solution containing 0.1% formic acid. After holding for 4 minutes, the acetonitrile concentration increased to 65% over 60 minutes.

FIG. 7A shows a diagram of the heated portion of an automated fluid peptide synthesizer. FIG. 7B shows a diastereomeric analysis of the model peptide GCF, showing a representative sample from fluid synthesis using Method B (top) and a 50/50 mixture of authentic Cys diastereomers (bottom). ing. FIG. 7C shows the percentage of Cys diastereomeric formation as a function of flow rate (ml / min) using Method B. FIG. 7D shows the same analysis as FIG. 7B for the model peptide FHL to investigate His epimerization during flow activation. LC-MS of the model peptide FHL (top panel) synthesized using Method B with a 50/50 mixture of authentic His diastereomers (bottom). FIG. 7E shows the percentage of histidine diastereomeric formation as a function of flow rate (ml / min).
(Example 5)

This example describes the materials, methods, and equipment configurations used in Example 4.

Materials: All reagents were purchased and used as received. The Fmoc amino acid was purchased from Creo Salus. Fmoc-His (Boc) -OH and O- (7-azobenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU) were purchased from ChemImpex. Omnisolv grade N, N-dimethylformamide (DMF) was purchased from EMD Millipore (DX1726-1). Diisopropylethylamine (DIEA, Catalog No. 387649), piperidine, trifluoroacetic acid, triisopropylsilane, acetonitrile, and 1,2-ethanedithiol (EDT) were purchased from Sigma Aldrich. The H-Rinkamide Chemmatrix polyethylene glycol resin was purchased from Pcas Biomatrix (Catalog No. 1744).

Reagent Storage and Fluid Manifold: The reagent storage system has two different tanks for accommodating reagents: a Chemglass 3-neck 500 mL spinner flask (CLS-1401-500) for large volumes, and a 50 mL polypropylene syringe tube (parts) for smaller volumes. # AD930-N, AD955-N) was used. All of the glass bottles were coated with UV resistant matte spray paint (Krylon 1309) to reduce UV degradation of the reagents, and these bottles had a green protective safety net for operation under argon pressure. The argon pressure was maintained at a pressure of 5 psi by a Swagelok pressure regulator (part # KCP1CFB2B7P60000). The reagent withdrawal line was equipped with a 20um polypropylene filter (part # JR-32178) to prevent the pump, check valve, and line from being clogged by crystallization or impurities of any reagent.

Each row of 9 amino acid bottles and syringes was fed to the VICI Valco 10 position valve (Vici part # C25-3180EUHA) and the 10th position was DMF. All of these valves were fed to the main Vici Valco 10 position valve. This main valve supplied the amino acid pump. Bottles containing HATU, other coupling agents, 40% piperidine, and DMF were fed to separate 10-position valves. This valve was connected to a coupling agent pump. DIEA is supplied directly to the third pump.

Pumping and mixing: AFPS operated with three Varian Prostar 210 pumps. The first pump delivered either amino acids or DMF. The second pump delivered either a coupling agent, a 40% piperidine solution, or DMF. The third pump delivered DIEA. The coupling agent and amino acid pump had a pump head of 50 ml / min (Agilent part # 50-WSS). The DIEA pump had a head of 5 ml / min (Agilent part # 5-Ti). The outlets of the three pumps meet at the cross section (IDEX component # P-722) with the three inlet check valves (IDEX component # CV-3320) to prevent diffusion between the cross section and the head. bottom. The lengths of the PEEK tubing (1/16 "OD, 0.020" ID) between the PEEK cross and all of the pumps had consistent volumes. A length of FEP tube (1/16 "OD, 0.030" ID) after the cross is coiled around a 1/2 inch cylinder 22 times to create a high Dean number (>). A static mixer of 3000) was formed to facilitate reagent mixing.

Activation and Coupling Reactor: After mixing, reagent flow proceeded to the selected heat exchanger using the VICI Valco 6-position column switching valve (Vici part # ACST6UW-EUTA). These heat exchangers consisted of a length of stainless steel pipe wrapped around an aluminum spool and coated with silicone for insulation. The spool was heated by two resistance cartridge heaters (Omega part # CSS-10250 / 120V). For peptide synthesis method A, a 3 m (10 ft, 1.368 ml) heat exchanger loop was used at 90 ° C; for peptide synthesis method B, a 1.5 m (5 ft, 0.684 ml) heat exchanger loop was used at 70 ° C. Used in.

Prototyping on Arduino: First, the control system was prototyped on Arduino Mega. Pumps and valves were daisy-chained and TTL serial ports were separated on the Arduino using RS232 MAX3232 SparkFun Transistor Breakout (BOB-11189).

Serial communication with pumps and valves: The standard RS-485 serial protocol was used to communicate with Varian ProStar 210 pumps and VICI Valco valves. Pump communication was 19200 baud, 8 bits, even parity, and 1 stop bit. The valve communication was 9600 baud, no parity, and 1 stop bit.

Heating and temperature control: All heaters were controlled by an 8-channel Waterow EZ-Zone RM controller (part number RMHF-1122-A1AA). This controller integrates PID control on the substrate. The temperature was read into the software through the RS-232 serial port using the software provided by Waterlow. All thermocouples were calibrated using one-point calibration at 0 degrees Celsius.

Process data acquisition: The software recorded temperature, mass flow rate, pressure, and UV absorbance during each synthesis. Temperature data was acquired using the Water PID control unit described above. For mass flow data, a Bronkhorst Coriolis mass flow meter (part # M14-XAD-11-0-5) was used to enable monitoring of fluid density. The differential pressure across the reactor was monitored using two DJ instrument HPLC through-hole titanium pressure sensors (part # DF2-01-TI-500-5V-41 "). These sensors were monitored at 100 psi. One point was calibrated at 90 degrees Celsius.

UV monitoring at 312 nm was achieved by using a Varian Prostar 230 UV-Vis ejector equipped with a Super Prep double path length flow cell (nominal path lengths of 4 mm and 0.15 mm). This dual path length flow cell device enables high dynamic range absorbance measurements-whenever the absorbance increases beyond the linear range of a large flow cell, the instrument switches to record the absorbance through a smaller flow cell. rice field. To ensure accurate measurements during flow cell switching, a route using a standard solution of dibenzofulvene prepared as described in Letters in Peptide Science, Vol. 9, pp. 203-206, 2002. The length ratio was calibrated.

Temperature and mass flow data were acquired through serial communication with the WaterpID and Bronkhorst flow meters. Voltage measurements for pressure and UV data were obtained from equipment using a National Instruments NI cDAC 9184 (part number 782069-01) equipped with a NI 9205 32-channel analog input card (part number 779357-01). Data points were recorded every 50 ms on average. In the UV detector, the signal response time was set to 10 ms and the total voltage scale was 100 mv.

The software allowed customization of amino acids, activators, coupling and deprotection step temperatures, coupling and deprotection step flow rates, and the amount of reagents used (number of pump strokes). These could be modified during operation of the system: for example, the temperature, the amount of amino acids used, or the activator could be changed in response to the UV that presented the aggregation.

The synthesizer was controlled via Ethernet® and USB on a Windows® computer with LabVIEW VI. The VI has a graphical interface that allows the user to easily create a recipe for the desired peptide. The recipe allows the user to control the flow rate, the amount of amino acids used, the activator, the temperature and residence time of activation, the deprotection residence time, and the amount of deprotection reagent at each step of synthesis. can. After creating the desired recipe, the user puts the recipe on the machine queue and presses "Run". During synthesis, the recipe can be modified in progress for any subsequent coupling step. When pressed "Run", the software will generate a predefined routine for each amino acid using the amino acid, flow rate, temperature, amount of reagent, and type of activation reagent selected by the user.

The cord consisted of operations performed by either a pump, a valve, or a motor. Each operation consisted of a set of inputs and pauses. The valve receives the valve ID and valve position; the pump receives the pump ID and pump flow rate; the motor receives the motor ID and motor position. After the step was completed, the program was kept waiting until the pause was completed before performing the next step. # Calculate the pause time represented by the variable during execution using recipe input. For example, the rest time after operating the pump in step 12 is determined by the "CPL NStrk" (number of coupling strokes) parameter in the recipe.

Analytical peptide cleavage and side chain protecting group removal: Approximately 10 mg of peptidyl resin was added to a 1.5 mL Eppendorf tube. 200 μL of the cleavage solution (94% TFA, 1% TIPS, 2.5% EDT, 2.5% water) was added to the tube and incubated at 60 ° C. for 5 minutes. After the cutting was completed, 200 μL TFA was added to the tube to rinse the resin and as much liquid as possible was transferred to another tube avoiding the resin using a pipette tip. 800 μL of cold diethyl ether was added to the tube of the cleavage solution. The tube was shaken-a visible waxy precipitate was formed and collected by centrifugation. The supernatant ether was poured, and two more ether washings were performed.

Finally, the waxy solid was briefly dried under a stream of nitrogen gas. 500 μL of a 50% aqueous acetonitrile solution was added to the tube and mixed thoroughly. This solution was filtered through a centrifuge basket filter, placed in a 50% aqueous acetonitrile solution containing 0.1% TFA, diluted 1:10, and subjected to liquid chromatography analysis.

Preparative peptide cleavage: After synthesis, the peptidyl resin was washed with dichloromethane, dried in a vacuum chamber and weighed. The resin was transferred to a 15 mL conical polypropylene tube. About 7 mL of cleavage solution (94% TFA, 1% TIPS, 2.5% EDT, 2.5% water) was added to the tube. More cutting solution was added to ensure complete immersion. The tubes were capped, inverted, mixed every half hour and allowed to proceed at room temperature for 2 hours.

The resin slurry was then filtered through a 10 μm polyethylene membrane disc fitted in a 10 mL Torviq syringe. The resin was rinsed twice more with 1 mL TFA and the filtrate was transferred to a 50 mL polypropylene conical tube. 35 mL of ice-cold diethyl ether was added to the filtrate and allowed to stand for 30 minutes to precipitate the peptide. The precipitate was collected by centrifugation and ground twice more with 35 mL of cold diethyl ether. Discarded the supernatant.

Finally, the residual ether was evaporated and the peptide was dissolved in a 50% aqueous acetonitrile solution. The peptide solution was frozen, lyophilized until dry and weighed.

Analysis of Peptide Samples Liquid Chromatographic Analysis: 1 μL of diluted peptide sample was analyzed on an Agilent 6520 LC-MS equipped with a Zorbax 300SB-C3 column (2.1 mm × 150 mm, 5 μm particle size). For the samples of FIGS. 6A-6C, a gradient of acetonitrile in water containing a 0.1% formic acid additive was used. The gradient started with 5% acetonitrile and increased to 65% acetonitrile at a rate of 1% acetonitrile per minute. The complete method included retention time at 1% along with the total time of the gradient.

Initial synthesis conditions and system characterization: At a total system flow rate of 20 mL / min and 70 ° C., treatment with 20% piperidine was selected to be 20 seconds, and the conditions shown above are sufficient for complete Fmoc removal. Was selected. DMF washing was selected to be 30 seconds. The wash time was verified using a UV detector to introduce Fmoc amino acids into the reactor and ensure that any UV active material was removed from the system after DMF wash.

The scheme for in-line mixing of activators and fluid streams of amino acids allowed a variety of conditions used for coupling. However, it required a deviation from the conditions traditionally used for aminoacylation in Fmoc synthesis. Typically, the reagents are used at their dissolution limit, about 0.4 M for Fmoc amino acids and uronium coupling agents. However, since these reagents are stored separately on AFPS and mix the two concentrated solutions in the coupling, the final solution initially used for aminoacylation consists of 0.2 M amino acids and an activator. Was done. In a typical coupling, a total of 9.6 mL of this coupling solution was used to ensure complete coupling. These conditions were first tested for the synthesis of the short polypeptide, ALFALFA.

Synthetic cycle optimization: ACP, a 10-residue peptide typically used as a diagnostic "difficult" sequence, with a total of 20, 40, and 60 mL / min using the same volume of coupling agent in each experiment. It was synthesized at a flow rate of 70 ° C. At higher flow rates, increased formation of chain stop by-products-increased tetramethylguanidyl cleavage during glutamine coupling was observed. This is hypothesized to be due to incomplete activation at high flow rates: there is residual HATU capable of guanidineylizing the N-terminus of the growing peptidyl chain when the amount of activator and amino acid is approximately equal. We were able to. By reducing the concentration of activator to 0.34M and ensuring complete head synchronization, this side reaction is almost always eliminated and ACPs are synthesized in quantitative yields at 80 mL / min. Will be possible. In Fmoc-Arg couplings in other peptides, these cleavages were still observed and therefore PyAOP was used as an activator for these couplings.

Investigation of the effect of temperature on deprotection: Fmoc-glycine functionalized peptidyl resin containing 20% piperidine was tested for deprotection at 70, 80, and 90 ° C. In all three cases, Fmoc-Gly was ligated to 200 mg of Chemmatrix Rinkamide resin at room temperature using a batch coupling method. The resin was then transferred to automated fluid synthesis and a single treatment of 20% piperidine was performed at either 70, 80, or 90 ° C. In all three cases, the integrated area of the Fmoc removal peak is the same, suggesting complete Fmoc removal. However, at higher temperatures, peak peaks occur early, suggesting faster deprotection and / or faster diffusion of the Fmoc-dibenzofulvene adduct leaving the resin.

Typical Protocol for Synthesizing Peptides with Automated Flow Peptide Synthesizers: Chemmatrix PEG Rinkamid Resin 200 mg, 6 mL Torviq with an additional 7-12 μm Polyx UHMWPE (XS-POR-7474) membrane on top of the frit. It was put into a frit syringe. The resin was pre-swelled with DMF for 5 minutes, after which the large resin agglomerates were manually destroyed by inserting a syringe plunger. The syringe was filled with DMF, charged into the fluid inlet, and charged into a heating chamber at 90 ° C. The synthesizer is set as shown in FIG. 5A and all reagents are pumped at a total flow rate of 80 mL / min, while the cross manifold, mixer and 10 ft stainless steel loop at 90 ° C. It is heated and then pumped onto the resin. Using three Varian Prostar 210 HPLC pumps, two had a 50 mL / min head for amino acids and activators, and one had a 5 mL / min lift for diisopropylethylamine (DIEA). .. A 50 mL / min lift pumped 400 μL of liquid per pump stroke; a 5 mL / min lift pumped 40 μL of liquid per pump stroke.

The standard synthetic cycle used included a first step of pre-washing the resin at 80 mL / min for 20 seconds at high temperature. During the coupling step, three HPLC pumps were used: a 50 mL / min lift pumped the activator (typically 0.34M HATU) and a second 50 mL / min lift was an amino acid. (0.4M) was pumped and a head of 5 mL / min delivered diisopropylethylamine (DIEA). The first two pumps started five pumping strokes to prime the coupling agent and amino acids before the DIEA pump was started. The three pumps were then operated together over a period of 7 pumping strokes, after which a rotary valve was used to switch between the activator pump and the amino acid pump so that DMF was selected. The three pumps were operated together over the final five pumping strokes, after which the DIEA pump was shut off and the other two pumps continued to wash the resin over an additional 16 pump strokes.

Two HPLC pumps were used during the deprotection step. By using a rotary valve, one HPLC pump selects 40% piperidine and the other selects DMF. The pump was operated over 13 pump strokes. After mixing, the final concentration of piperidine is 20%. The rotary valve then selected DMF for both HPLC pumps and the resin was washed over an additional 16 pump strokes. The coupling / deprotection cycle was repeated for all additional monomers.

Aspartimid formation and high temperature GHRH synthesis: GHRH synthesis at 70 ° C and 90 ° C was investigated. When this synthesis was performed at 90 ° C, in contrast to 70 ° C, formation of aspartimid by-products with sine-18Da mass and shifted residence time was observed. This side reaction is known to occur at both high temperatures and certain Asp-containing peptides. The effects of piperazine, a more mild base, on this side reaction were investigated. By using 2.5% piperazine instead of 20% piperidine for deprotection, the amount of this by-product was significantly reduced when measured by LC-MS, but the amount of amino acid deletion was increased. And especially for Ala and Leu. The addition of 0.1 M HOBt to the 2.5% piperazine deprotected cocktail resulted in almost the same synthetic quality. Therefore, for ASP-containing peptides suspected of aspartimid formation, it is advantageous to use either or both of the reduced temperature, reduced intensity deprotection cocktails.

Manual Synthesis of Insulin B Chain and GHRH: These peptides were synthesized according to Kent et al., Org Lett. 2015, Vol. 17, Vol. 14, p. 3521. A Chemmatrix Rink-amide resin (0.1 mmol; 0.45 mmol / g) was used. First, 0.55 mmol of the amino acid to be coupled was dissolved in 1.25 mL of 0.4 M HBTU / 0.4 M HOBT, and then 122 μL (0.7 mmol) of DIEA was added to activate the amino acid for 30 seconds. After 30 seconds, the solution was added to the resin. The coupling was allowed to proceed for 30 minutes with intermittent stirring.

After each coupling step, 45 mL of DMF flow washes were performed. 3 mL of 20% (v / v) piperidine was then added to the resin, stirred and incubated for 5 minutes. This process was repeated once. After each deprotection, 45 mL of fluid washes were performed, followed by 1 minute batch treatment with DMF.

Determination of Cys and His epimerization: Cys and His epimerization were measured using two model peptides GCF and FHL, respectively. For each synthesis, the flow rates for C and H couplings were varied and the coupling conditions for flanking residues (G and F for GCF; F and L for FHL) were set at 90 ° C. and 80 mL / min. The total flow rate was kept constant. After the synthesis of each model peptide, cleavage was performed as described above.

LC-MS analysis of the truncated product was performed. Extracted ion chromatograms of the two stereoisomers were obtained to determine the amount of D-epimer formed in each case: 342.5 to 329.0 Da for GCF, and 494 for FHL. 9 to 417.6 Da. The peaks corresponding to each epimer were integrated. Authentic standards were prepared and analyzed in the same way to verify the residence time of each epimer.

Although some embodiments of the invention have been described and exemplified herein, one of ordinary skill in the art will exercise and / or obtain results and / or one or more of the advantages described herein. Various other means and / or structures for this will be readily imagined, and each such variant and / or modification is considered to be within the scope of the invention. More generally, one of ordinary skill in the art will mean that all parameters, dimensions, materials, and configurations described herein are exemplary, with actual parameters, dimensions, materials, and / or configurations being used. , It will be readily appreciated that the teachings of the present invention (s) will depend on one or more specific applications in which they are used. One of ordinary skill in the art will be able to recognize or confirm many equivalents of the particular embodiments of the invention described herein using only conventional experiments. Accordingly, the aforementioned embodiments are provided by way of example only, and the invention may be practiced within the appended claims and its equivalents, in addition to those specifically described and claimed. Please understand that it is good. The present invention is directed to each of the individual features, systems, articles, materials, and / or methods described herein. Moreover, any combination of two or more such features, systems, articles, materials, and / or methods are mutually inconsistent with such features, systems, articles, materials, and / or methods. If not, it is included within the scope of the present invention.

As used herein and in the claims, the indefinite articles "one (a)" and "one (an)" are understood to mean "at least one" unless the opposite is explicitly stated. Should be.

It should be understood that the word "and / or" as used herein and in the claims means "either or both" of such associated elements, i.e. the element is the case. Some exist jointly, others detachably. Other elements, whether related or unrelated to those specifically specified, are optional except for those specifically specified by the "and / or" clause, unless the opposite is explicitly stated. It may exist. Thus, as a non-limiting example, when the term "A and / or B" is used in conjunction with a non-limiting language such as "contains", in one embodiment A without B (optionally). , Including elements other than B); in another embodiment B without A (optional, including elements other than A); in yet another embodiment both A and B (optional, other elements) Including) and so on.

As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., a list of several elements or elements, and an optional additional enumeration. It shall include at least one of the items not listed, but more than one. Only terms that specify the opposite, such as "only one of" or "exactly one of", or "consisting of" when used in the claims, are some elements or It means to include exactly one element in the list of elements. In general, the term "or" as used herein is an exclusivity term such as "any", "one of", "only one of", or "exactly one of". When preceded, it shall simply be construed as indicating an exclusive alternative (ie, "one or the other but not both"). "Being essential from" shall have its usual meaning as used in the field of patent law when used in the claims.

As used herein and in the claims, the phrase "at least one" is selected from any one or more of the elements in the list of elements when referring to the list of one or more elements. Means at least one element, but understands that it does not necessarily include at least one of each and all of the elements specifically listed in the list of elements and does not exclude any combination of elements in the list of elements. Should be. According to this definition, an element may or may not be specifically related to the specified element, except for the specifically specified element in the list of elements pointed to by the word "at least one". It is also possible that it may exist in. Thus, as a non-limiting example, "at least one of A and B" (or equally "at least one of A or B", or equally "at least one of A and / or B") is an embodiment. In the case, at least one A containing more than one in the arbitrary choice and B does not exist (in the optional choice, the elements other than B are included); in another embodiment, the optional choice contains more than one. At least one B containing many and no A (optional, including elements other than A); in yet another embodiment, with at least one A containing more than one in the optional. , At least one B (arbitrarily optional, including other elements) containing more than one in the optional option.

In the claims, and in the specification above, "comprising," "including," "carrying," "having," "containing," and "involved." It should be understood that all transitional phrases such as "involving" and "holding" are understood to be open-ended, i.e., meaning include but not limited to them. Only the transitional phrases "consisting of" and "consisting essentially of" are described in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It shall be a closed or semi-closed transition clause, respectively.

Claims (30)

A way to initiate the operation of a peptide synthesis system,
Initiating a first fluid stream containing amino acids from the first reagent reservoir into the mixing region,
The second fluid flow containing a base is started to flow from the second reagent reservoir to the mixed region, and the leading edge of the first fluid flow and the leading edge of the second fluid flow are within 25 ms of each other. To reach the mixed region,
A method of merging the first and second fluid streams in the mixing region, comprising forming a mixed fluid stream and allowing the mixed fluid stream to flow into a reactor. A way to initiate the operation of a peptide synthesis system,
Initiating a first fluid stream containing an activator from the first reagent reservoir into the mixing region,
The second fluid flow containing a base is started to flow from the second reagent reservoir to the mixed region, and the leading edge of the first fluid flow and the leading edge of the second fluid flow are within 25 ms of each other. To reach the mixed region,
A method of merging the first and second fluid streams in the mixing region, comprising forming a mixed fluid stream and allowing the mixed fluid stream to flow into a reactor. A way to initiate the operation of a peptide synthesis system,
Initiating a first fluid stream containing amino acids from the first reagent reservoir into the mixing region,
A second fluid stream containing an activator is initiated from the second reagent reservoir into the mixing region, wherein the leading edge of the first fluid stream and the leading edge of the second fluid stream are mutually exclusive. To reach the mixed region within 25 ms,
A method of merging the first and second fluid streams in the mixing region, comprising forming a mixed fluid stream and allowing the mixed fluid stream to flow into a reactor. The method of claim 1, comprising flowing a third fluid stream containing an activator. The method of claim 2, comprising flowing a third fluid stream containing an amino acid. The method according to any one of claims 4 to 5, further comprising merging the first, second, and third fluid streams in the mixed region. The method of any one of claims 4-6, comprising flowing a fourth fluid stream comprising an additive selected from the group consisting of chaotropic salts, co-solvents, and surfactants. The method of claim 7, further comprising merging the first, second, third, and fourth fluid streams in the mixing region. The method according to any one of claims 2 to 8 , wherein the activator is selected from the group consisting of carbodiimide, guanidinium salt, phosphonium salt, and uronium salt. The method according to any one of claims 1 to 9 , wherein the leading edge of the first fluid flow reaches the mixed region within 10 ms from the leading edge of the second fluid flow. The method according to any one of claims 1 to 2 and 4 to 10 , wherein the base is a Lewis base. The method according to any one of claims 1 to 2 and 4 to 10 , wherein the base is a non-nucleophilic base. The method according to any one of claims 1 to 12 , wherein the mixed fluid flow is not exposed to heat from a heat source before reaching the reactor. The method according to any one of claims 1 to 13 , wherein the mixed fluid flow is not exposed to heat from a heat source between the mixing region and the entrance to the reactor. The method according to any one of claims 1 to 14 , wherein the temperature of the mixed fluid flow is within 10 ° C. of the temperature of the leading edge at the entrance of the reactor. The method according to any one of claims 1 to 15 , wherein the temperature of the leading edge changes by less than 10 ° C. from the mixing region to the inlet of the reactor. The method according to any one of claims 1 to 16 , wherein the reactor does not contain a plurality of amino acids immobilized on a solid support. The method according to any one of claims 1 to 17 , wherein the reactor does not contain a plurality of peptides immobilized on a solid support. The method according to any one of claims 1 to 16 , wherein the reactor comprises a plurality of peptides immobilized on a solid support. 19. The method of claim 19 , comprising exposing the amino acid to the immobilized peptide, wherein at least a portion of the amino acid is bound to the immobilized peptide to form a long peptide. 20. The method of claim 20 , wherein more than about 99% of each of the immobilized peptides is bound to a single amino acid molecule during the exposure step, respectively. The method of any one of claims 19-21 , wherein the solid support is housed in a packed column and / or a fluidized bed. The method according to any one of claims 19 to 22 , wherein the solid support contains a resin. The method according to any one of claims 19 to 23 , wherein the solid support comprises a microporous polystyrene resin, a microporous polyethylene glycol resin, and / or a microporous copolymer resin. The method of any one of claims 1-16 and 19-24 , further comprising providing a plurality of peptides comprising a protecting group, each peptide immobilized on a solid support. Claiming that exposing the deprotecting reagent to the immobilized peptide further comprises removing the protecting group from at least a portion of the immobilized peptide and removing at least a portion of the deprotecting reagent. Item 25 . Exposing the activated amino acid to the immobilized peptide further comprises forming a newly bound amino acid residue by binding at least a portion of the activated amino acid to the immobilized peptide. The method according to any one of claims 19 to 26 . 27. The method of claim 27 , further comprising removing at least a portion of the activated amino acid that does not bind to the immobilized peptide. The method according to any one of claims 25 to 26 , wherein the protecting group comprises a fluorenylmethyloxycarbonyl protecting group. The method of any one of claims 25-26 , wherein the protecting group comprises a tert-butyloxycarbonyl protecting group.

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